Genetic Engineering of Mammalian Sensory Circuits

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Optogenetics has emerged as a powerful tool to control neuronal activity with high spatio-temporal precision. We were the first to develop optogenetic strategies to activate selectively genetically-defined peripheral somatosensory neurons involved in nociception and pain in mice (Daou et al., 2013). In this 5-year research program, we propose to design and validate conditional transgenic models for the functional interrogation of neuronal subsets with unclear roles in somatosensory circuits.Our main scientific objectives are: 1) To interrogate genetically-identified neurons in somatosensory circuits in vivo using optogenetics. Primary sensory neurons are located in the dorsal root, trigeminal, and nodose ganglia while their peripheral terminals and fibers innervate the skin and peripheral tissues. Their activation is directly linked to somatosensory modalities (touch, temperature, itch, nociception) and to pain perception. We previously used the "proof-of-concept" Nav1.8-Cre driver with broad neuronal expression but we propose now to interrogate more specific subsets of genetically-defined neurons: thermal peptidergic nociceptors with TRPV1-Cre, tactile non-peptidergic nociceptors with MrgD-Cre, itch sensors with MrgA3-Cre and mechanosensors with TH-Cre. Once their optical activation with ChR2 or inhibition with Arch is demonstrated in vitro and in vivo behaviorally in normal conditions (tactile response, thermal sensitivity, conditioned place aversion), we will test their contribution to chronic pain following inflammation or neuropathic injury using behavioral analysis and quantitative sensory measurements. 2) To modulate selectively the two main types of nociceptors in vivo using chemogenetics. Peripheral sensitization of nociceptors plays a major role in triggering central mechanisms of chronic pain. We have shown that TRPC3-linked Gq-coupled pathways contribute to peripheral sensitization in nociceptors (Alkhani et al., 2014). Using AAV viral expression of two chemogenetic actuators, the DREADDs M3D (Gq-coupled) and M4D (Gi-coupled) activated by the compound CNO, we propose to investigate the functional consequences of CNO-induced modulation of TRPV1+ and MrgD+ nociceptor firing in transgenic mice in vitro and in vivo. We make the hypothesis that activation of M3D Gq-coupled pathways will promote sensitization and inflammation, while M4D Gi-coupled pathways will protect DRG/TG neurons from sensitization and hyperexcitability, recruiting cellular mechanisms analogous to morphine analgesia. These novel transgenic mice engineered with artificial peripheral transducers/receptors will allow us to dissect in vitro the signaling diversity of sensory neurons as well as to investigate in vivo the contribution of specific neuronal populations to several modalities of somatosensation relevant to pain, in normal and sensitized states.